Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons

<p>Abstract</p> <p>Background</p> <p>Chloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl^-accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl^-currents. The intracellular Cl^-concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl^-currents to signal generation differs between individual afferent neurons, and whether the specific Cl^-levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl^-homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl^-concentrations and on the expression levels of Cl^-transporters in rat DRG neurons.</p> <p>Results</p> <p>We developed an <it>in vitro </it>assay for testing how inflammatory mediators influence Cl^-concentration and the expression of Cl^-transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 μM ATP, 0.9 μM bradykinin, and 1.4 μM PGE₂for 1–3 hours. Two-photon fluorescence lifetime imaging with the Cl^--sensitive dye MQAE revealed an increase of the intracellular Cl^-concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na⁺-K⁺-2Cl^-cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl^-levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl^-importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl^-homeostasis toward a state of augmented Cl^-accumulation, which is induced by a 1–3 hour treatment with inflammatory mediators.</p> <p>Conclusion</p> <p>Our findings indicate that inflammatory mediators impact on Cl^-homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl^-levels and, hence, the driving force for depolarizing Cl^-efflux. These findings corroborate current concepts for the role of Cl^-regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl^-concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl^-currents in the presynaptic terminals. Moreover, excitatory Cl^-currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.</p

In Vitro Effect of 9,9′-Norharmane Dimer against Herpes Simplex Viruses

Herpes simplex virus (HSV) infections are highly widespread among humans, producingsymptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threateningencephalitis. At present, there are no vaccines available, and some existing antiviral treatments canbe ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In thisreport, the in vitro anti-HSV effect of 9,9′-norharmane dimer (nHo-dimer), which belongs to the β-carboline (βC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and didnot impede either the aachment or penetration steps of viral particles. The antiviral effect was onlyexerted under the constant presence of the dimer in the incubation media, and the mechanism ofaction was found to involve later events of virus infection. Analysis of fluorescence lifetime imagingdata showed that the nHo-dimer internalized well into the cells when present in the extracellularincubation medium, with a preferential accumulation into perinuclear organelles including mitochondria.After washing the host cells with fresh medium free of nHo-dimer, the signal decreased,suggesting the partial release of the compound from the cells. This agrees with the observation thatthe antiviral effect is solely manifested when the alkaloid is consistently present in the incubationmedia.Fil: Gonzalez, Maria Micaela. Universidad Nacional de San Martin. Instituto Tecnologico de Chascomus. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - la Plata. Instituto Tecnologico de Chascomus.; ArgentinaFil: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Erra Balsells, Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Gensch, Thomas. Helmholtz Gemeinschaft. Forschungszentrum Jülich; AlemaniaFil: Cabrerizo, Franco Martín. Universidad Nacional de San Martin. Instituto Tecnologico de Chascomus. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - la Plata. Instituto Tecnologico de Chascomus.; Argentin

Automatic Bayesian single molecule identification for localization microscopy

Single molecule localization microscopy (SMLM) is on its way to become a mainstream imaging technique in the life sciences. However, analysis of SMLM data is biased by user provided subjective parameters required by the analysis software. To remove this human bias we introduce here the Auto-Bayes method that executes the analysis of SMLM data automatically. We demonstrate the success of the method using the photoelectron count of an emitter as selection characteristic. Moreover, the principle can be used for any characteristic that is bimodally distributed with respect to false and true emitters. The method also allows generation of an emitter reliability map for estimating quality of SMLM-based structures. The potential of the Auto-Bayes method is shown by the fact that our first basic implementation was able to outperform all software packages that were compared in the ISBI online challenge in 2015, with respect to molecule detection (Jaccard index)